ABSTRACT: Aanlysis of human resting CD4+T cells and cells activated by two different methods: CD4+ T cells unstimulated (be susceptible but not support to HIV-1) and stimulated either by CD3/CD28 costimulation (reversed susceptibility and resisted to HIV-1) or by PHA/IL-2 for six days (be susceptible and support to HIV-1) to investigate potential mechanism of reversing susceptible to HIV-1. We measured gene expression in resting human CD4+T cells and cells activated by two methods for six days which could induce different effects to HIV-1. Three independent experiments were performed at each treatments using different donors for each experiment.
Project description:Memory T cells (TM) play a prominent role in protection and auto-immunity due to their ability to mount a more effective response than naïve T cells (TN). However, the molecular mechanisms underlying enhanced functionality of TM are not well defined, particularly in human TM. We examined the global gene expression profiles of human CD8+ TN and TM before and after stimulation. There were 1,284, 1,373 and 1,629 differentially expressed genes between TN and TM at 0 hr, 4 hr and 24 hr after stimulation, respectively, with more genes expressed to higher levels in TM. Genes rapidly up-regulated in TN cells were largely involved in nitrogen, nucleoside and amino acid metabolisms. In contrast, those in CD8+ TM were significantly enriched for immune-response-associated processes, including cytokine production, lymphocyte activation and chemotaxis. Multiple cytokines were rapidly up-regulated in TM cells, including effector cytokines known to be produced by CD8+ T cells and important for their functions, as well as regulatory cytokines, both pro- and anti-inflammatory, that are not typically produced by CD8+ T cells. These results provide new insights into molecular mechanisms that contribute to the enhanced functionality of human CD8+ TM and their prominent role in protection and auto-immunity. Naïve and memory phenotype CD8 T cells were purified by FACS from healthy individuals and cultured in vitro with the stimulation of anti-CD3/CD28 mAbs for 0 hr, 4 hr and 24 hr. Total RNA was purified from un-stimulated and stimulated naive and memory CD8 T cells and hybridized to individual single-color arrays.The purification and stimulation protocol was performed two independent times.
Project description:Expanding the sequencing depth of the peptides showing a statistically significant quantitative change arising from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system. A fritless, 50 cm-long column packed with 1.9 µm particles operated with a standard pressure HPLC significantly improved the sequencing depth 51% and decreased the selected ion chromatogram peak spreading. Most importantly, under the optimal workflow we observed an improvement of 330% in detection of significantly changed phosphopeptides in the stimulated cells compared with the other workflows. The discovery power of the optimized column configuration was illustrated by identification of significantly altered phosphopeptides harboring novel sites from proteins previously established as important in T cell signaling including A-Raf, B-Raf, c-Myc, CARMA1, Fyn, ITK, LAT, NFAT1/2/3, PKCα, PLCγ1/2, RAF1, and SOS1. Taken together, our results revealed a simple column fabrication methodology that provides an inexpensive improvement for single-run LC-MS/MS analysis to optimize peptide sequencing depth, dynamic range, sensitivity, and label free quantitative reproducibility.
Project description:The purpose of this study was to determine which genes are differentially regulated by the E3 ligase Nrdp1 in CD8+ T cells after treatments with anti-CD3/CD28 Abs. The results demonstrate increased induction of cytotoxicity-associated genes in Nrdp1-/- mice than in Nrdp1+/+ mice after activation. Thus Nrdp1 may be involved in the regulation of TCR signaling. Naive CD8+ T cells derived spleens of Nrdp1+/+ and Nrdp1-/- mice were either untreated or treated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for 4h or 8h. Equal amounts of RNA were assayed for gene expression using Affymetrix mouse 430 2.0 arrays.
Project description:Predose and postdose gene expression profiles of blood samples of five most susceptible and five most resistant rats to acetaminophen-induced hapatotoxicity were determined by microarray analysis. Four out of ten selected genes were found to be reproducibly in real-time PCR with independent set animals to predict the susceptible individuals to acetaminophen-induced hapatotoxicity. We combined gene expression profiles of predose and postdose rat blood to predict the susceptible individuals to acetaminophen-induced hapatotoxicity.
Project description:Mouse splenic dendritic cells are divided into different subsets based on their phynotype. CD8α+ and CD8α- dendritic cells play different roles against pathogens. Cross-presentation is essential for immune defense against viruses, tumors and intracellular bacteria and CD8α+ DCs are more potent in cross-presentation compared to CD8α- DCs. We used microarrays to detail the global gene expression to analyze the underlying mechanism in CD8α+ dendritic cells cross-presentation Splenic CD8α+ and CD8α- dendritic cells were sorted by flow-cytometer for RNA extraction and hybridization on Affymetrix microarrays. Thirty C57BL/6J mice were sacrified for the splenic cells, splenic DCs were enriched by negtive selection, then CD8α+ and CD8α- dendritic cells were seprated based on CD11c and CD8α markers.
Project description:T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into the Th1, Th2, Th17 or iTreg phenotype, depending on environmental co-stimulatory signals. In order to identify the genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli an pathway inhibitors Jurkat T cells were treated with CD3, CD28 and PMA and all pairwise combinations, in the presence of DMSO (control) or kinase inhibitors. RNA was isolated after 8 hrs incubation.
Project description:T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into the Th1, Th2, Th17 or iTreg phenotype, depending on environmental co-stimulatory signals. In order to identify the genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli an pathway inhibitors Jurkat T cells were treated with CD3/CD28 and CD3/PMA and CD28/PMA. RNA was isolated after 1 and 8 hrs stumalation.
Project description:Serum is a valuable body fluid to diagnose cancer as it can be accessed with minimal invasive techniques. Studying the cancer serum proteome provides valuable insights into the pathophysiology of tumor progression. Gastric adenocarcinoma is an aggressive cancer resulting in poor prognosis, mainly due to the lack of specific early diagnostic biomarkers. To this end, we used an iTRAQ-based quantitative proteomic approach to identify differentially expressed proteins in the sera of patients diagnosed with gastric cancer. Our study resulted in the identification of 643 proteins in the serum, of which 48 proteins were found to be overexpressed and 11 proteins underexpressed in gastric cancer when compared with healthy controls. We used multiple reaction monitoring assays to validate the overexpression of potential biomarkers. This catalog of serum-based biomarkers will aid in diagnosis and prognosis of gastric cancer.
Project description:The relationship between host microRNAs (miRNA), viral control and immune response has not been elucidated in the field of HIV yet. The aim of this study was to assess the differential miRNA profile in CD8+ T-cells between HIV-infected individuals who differ in terms of disease progression. A total of 136 RNAs (miRNAs) were analyzed: 68 RNAs from resting CD8+ T-cells and the respective 68 RNAs from stimulated CD8+ T-cells from Elite Controllers (EC, n=15), Viremic Controllers (VC, n=15), Viremic Progressors (VP, n=13), Treated Patients (ART, n=14) and Uninfect Donors (HIV-, n=11).
Project description:To identify the differentially expressed genes in metallopanstimulin-1 (MPS-1) knockdown gastric cancer cells compared with negative control ones, we employed the microarray profiling analysis. MPS-1 was knockdown by retroviral interfering system in human gastric adenocarcinoma originated cell SGC7901 and the transfectants named P4, while the negative control named NC. Genes with greater than 1.5-fold change and P-value ＜0.05 were identified as differentially expressed genes between NC and P4 cells. Among those, apoptotic related gene (Gadd45β, cIAP2, Bcl2, CAD, Bid, etc) and adhesive related genes (integrin beta 4, ECM2, etc) were quantified by real-time PCR as well as western blotting. The two groups of negative control (NC) and MPS-1 knockdown gastric cancer cells (P4) were harvested after puromycin screening. Three independent experiments were performed for each group.